Myc proteins are essential for normal cellular function and embryonic development. However, when its normal regulation is compromised, Myc acts as a major contributor to the initiation and progression of a wide range of human cancers. Myc has been long known to be a transcription factor that heterodimerizes with the Max protein. The Myc-Max dimer binds DNA and regulates expression of thousands of genes involved in cell growth and proliferation. The long-term objectives of this grant application, as embodied in our three Specific Aims, are to elucidate two previously unexplored tumor-specific pathways through which Myc drives neoplasia and to devise an approach to specifically inhibit Myc activity. In Aim 1 we extend our recent discovery of a transcriptionally inactive form of Myc which promotes survival and motility of cancer cells. We have shown that Myc protein is proteolytically cleaved to generate a cytoplasmic, transcriptionally inactive, N-terminal segment of Myc, denoted as Myc-nick. Myc-nick is produced in response to growth arrest or stress, and is present in a wide range of human tumors. Through its ability to interact with acetyltransferases, Myc-nick acetylates cytoplasmic proteins, alters cell morphology, promotes survival and stimulates filopodia formation and cell motility. We propose experiments, using tumor models in Zebrafish and mice, to determine the involvement of Myc-nick in tumor progression and metastasis. In addition, we will employ proteomics to identify Myc-nick interacting proteins and the molecular basis for Myc- nick's functions. Aim 2 is focused on a gain of oncogenic function caused by a point mutation in Myc. This coding region mutation (Myc-T58A), which occurs in the conserved Myc phospho-degron (Myc Box I), accelerates oncogenicity of overexpressed Myc. To separate the effects of the T58A mutation from the effects of Myc overexpression, we generated mice containing T58A in the endogenous myc locus. In these mice Myc- T58A is normally regulated and there is no overt increase in tissue growth or proliferation. Yet c-myc-T58A mice display increased hematopoietic progenitor cell self-renewal and exhibit a long-latency tumor-prone phenotype. We surmise that the Myc-T58A mutation alters a protein interaction with Myc Box I and influences activation of a subpopulation of Myc target genes. We plan to identify the basis for the T58A phenotypes using proteomic and genomic analyses. In Aim 3 we propose to generate highly specific peptide-based inhibitors of Myc-Max dimerization using binding selection of ~104 Max variants coupled with high-throughput sequencing. Peptides that inhibit Max dimerization with Myc, but not with Mxd repressors, will be examined for their ability to inhibit growth of Myc-driven tumors. This general approach can be applied to other critical protein interactions. We anticipate that the studies proposed in this application will lead to new insights into Myc's activities in cancer and to novel approaches aimed at inhibiting the function of this critical oncogene.